Spinal cholinergic interneurons regulate the excitability of motoneurons during locomotion -- Miles et al. 104 (7): 2448 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Miles et al. 10.1073/pnas.0611134104.

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Fig. 5. C boutons do not originate from motoneurons. (A) Fluorescent (´25) images of GFP, ChAT, and FluoroGold-labeled lumbar spinal MNs in a ChAT-GFP transgenic mouse obtained from the Gensat project. In these animals, GFP expression was observed in all MNs (n = 2; 9 sections in one and 10 in the other). (B) Confocal images (´63) showing Renshaw cells identified by their location and calbindin immunoreactivity [Carr PA, Alvarez FJ, Leman EA, Fyffe RE (1998) NeuroReport 9:2657-2661], surrounded by vesicular acetylcholine transporter (VAChT) and GFP-labeled terminals (projections of six single optical sections, obtained at 1-mm intervals; arrowheads indicate VAChT⁺/GFP⁺ terminals). (C) High-power (´63) confocal images showing a lumbar spinal MN labeled with GFP and surrounding GFP^- C boutons labeled for VAChT (projections of 10 single optical sections, obtained at 1-mm intervals). Given that the C boutons are GFP^- (C) and the central terminals of motoneurons are GFP⁺ (B), C boutons do not arise from MNs. (D) Low-power (´10) confocal images showing immunolabeling for ChAT and GFP in the lumbar spinal cord (projection of 24 single optical sections, obtained at 2-mm intervals). Some cholinergic neurons are GFP^- (arrows), and thus a subset of these must give rise to the C bouton input to MNs. (Scale bars: A, 100 mm; B, 10 mm; C, 50 mm; D, 10 mm.) For details, see SI Methods.

SI Methods

ChAT-GFP mice (1) were anaesthetized and perfused with 4% paraformaldehyde. Transverse 50-mm spinal cord sections were cut with a freezing microtome and incubated for 16 h in combinations of the following: rabbit anti-GFP (1:1,000; Chemicon, Temecula, CA), rabbit anti-ChAT (1:100; Chemicon), goat anti-vesicular acetylcholine transporter (VAChT; 1:5,000; Chemicon), and mouse anti-calbindin (1:5,000; Swant, Switzerland). They then were incubated for 2 h in appropriate donkey secondary antibodies (1:250; Invitrogen, Burlington, ON) conjugated to Alexa 488, Alexa 550, or Alexa 647. Confocal images were acquired with a Zeiss LSM 510 Meta confocal microscope.

1. Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N (2003) Nature 425:917-925.